THE ANTIBODY SPECIFIC FOR MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE PHOSPHORYLATED BY PROTEIN-KINASE-C - ACTIVATION OF PROTEIN-KINASE-CIN SMOOTH-MUSCLE CELLS IN HUMAN CORONARY-ARTERIES

Myristoylated alanine-rich C kinase substrate (MARCKS), a major substr ate for protein kinase C, is distributed in a variety of cells. It has been reported that phosphorylation of MARCKS at serines 152 and 156 a ccording to the numbering of rat brain MARCKS can be used as an indica tor for protein kinase C activation in intact cells. To detect the act ivation of protein kinase C in vivo, we produced a specific antibody a gainst MARCKS phosphorylated at serines 152 and 156. We synthesized a phosphopeptide which contained phosphoserines 152 and 156 and prepared the antibody specific for this phosphopeptide. Immunoblot analysis wi th both purified MARCKS and the cytosol fraction from rat brain reveal ed that the antibody reacted only with MARCKS phosphorylated by protei n kinase C, The antibody was suitable for immunoblot analysis and immu nostaining with cultured human coronary artery smooth muscle cells. Ph osphorylation of MARCKS was increased about eightfold by the treatment of the cells with phorbol 12-myristate 13-acetate, a protein kinase C activator. Furthermore, treatment of the cells with endothelin-l and tumor necrosis factor alpha increased phosphorylation of MARCKS, Inter estingly, phosphorylation of MARCKS was clearly observed in smooth mus cle cells in atherosclerotic lesion of subjects at autopsy. These resu lts suggest that the antibody is useful for examination of the activat ion of protein kinase C in vascular smooth muscle cells in vivo. (C) 1 998 Academic Press.